Role of indoleamine 2,3-dioxygenase in testicular immune-privilege

Male meiotic germ cell including the spermatozoa represent a great challenge to the immune system, as they appear long after the establishment of normal immune tolerance mechanisms. The capacity of the testes to tolerate autoantigenic germ cells as well as survival of allogeneic organ engrafted in the testicular interstitium have led to consider the testis an immunologically privileged site. Disruption of this immune privilege following trauma, tumor, or autoimmune orchitis often results in male infertility. Strong evidence indicates that indoleamine 2,3-dioxygenase (IDO) has been implicated in fetal and allograft tolerance, tumor immune resistance, and regulation of autoimmune diseases. IDO and tryptophan 2,3-dioxygenase (TDO) catalyze the same rate-limiting step of tryptophan metabolism along a common pathway, which leads to tryptophan starvation and generation of catabolites collectively known as kynurenines. However, the relevance of tryptophan metabolism in testis pathophysiology has not yet been explored. Here we assessed the in vivo role of IDO/TDO in experimental autoimmune orchitis (EAO), a model of autoimmune testicular inflammation and immunologically impaired spermatogenesis. EAO was induced in adult Wistar rats with testicular homogenate and adjuvants. Control (C) rats injected with saline and adjuvants and normal untreated rats (N) were also studied. mRNA expression of IDO decreased in whole testes and in isolated Sertoli cells during EAO. TDO and IDO localization and level of expression in the testis were analyzed by immunostaining and Western blot. TDO is expressed in granulomas from EAO rats, and similar protein levels were observed in N, C, and EAO groups. IDO was detected in mononuclear and endothelial cells and reduced IDO expression was detected in EAO group compared to N and C rats. This phenomenon was concomitant with a significant reduction of IDO activity in EAO testis measured by tryptophan and kynurenine concentrations (HPLC). Finally, in vivo inhibition of IDO with 1-methyl-tryptophan increased severity of the disease, demonstrating down regulation of IDO-based tolerance when testicular immune regulation was disrupted. We present evidence that an IDO-based mechanism is involved in testicular immune privilege.


Induction of experimental autoimmune orchitis (EAO).
Rats were actively immunized with syngeneic testicular homogenate (TH) emulsified with complete Freund's adjuvant (CFA; Sigma-Aldrich, St Louis, MO, USA) into the hind footpads and in different sites of flanks and near the neck area (subcutaneously) as described previously 26 . The first two immunizations were followed by an intravenous injection of 10 10 inactivated Bordetella pertussis (Bp) bacteria (strain 10536; kindly provided by Instituto Malbrán, Buenos Aires, Argentina), whereas the third was followed by an intraperitoneal injection of Bp at a concentration of 5 × 10 9 . Rats of control (C) group received CFA and Bp, but no TH, otherwise following the same scheme. Normal (N) untreated rats were also studied. Rats were killed 50 days after the first immunization. Testes, epididymis and popliteal, inguinal, renal and iliac lymph nodes (LN) were removed, weighed, and processed as described below. Only rats developing orchitis after 50 days were studied (EAO rats).
Histopathology. Histopathology of testis and epididymis was evaluated in paraffin-embedded Bouin's-fixed sections obtained from three different levels and stained with hematoxylin-eosin. To evaluate the severity of EAO, we used a score described previously 27 . This score was graded by evaluation of (a) percentage of ST with impairment of spermatogenesis, (b) degree of germ cell sloughing and (c) testicular/body weight ratio (T/Bw). Maximum EAO score is 10. Animals with a score equal to 0 were considered free of orchitis. Epididymal pathology was graded by evaluation of caput, corpus, and cauda inflammation and sperm depletion using an established score 28 . Epididymal inflammation (epididymitis): 1-5 and sperm depletion: 0-2 represent the range of incremental inflammation and decrement of sperm, respectively.
Immunohistochemistry. Rabbit polyclonal antibodies anti-IDO that recognize at least rat and human IDO1 29 or anti-TDO (GeneTex, Irvine, CA, USA) were used to detect expression and localization of IDO and TDO proteins in acetone-fixed frozen testis and LN sections (7 µm thick). Sections were incubated with 5% normal goat serum, 0.03% Triton X-100 containing 4% bovine serum albumin (BSA) for IDO or with 5% skim milk and 0.01% Triton X-100 for TDO 30 min at room temperature, and treated with avidin/biotin blocking solution (Vector Lab., Burlingame, CA, USA) followed by overnight incubation with the primary antibody IDO (1/500) or TDO (1/25) at 4 °C in a humidified chamber. A biotinylated goat anti-rabbit IgG (1/250, Vector Lab.) was used as secondary antibody. Endogenous peroxidase activity was blocked by treatment with 0.01% H 2 O 2 in methanol for 30 min. The reaction was amplified with the Vectastain Elite ABC Kit (Vector Lab.), and the reaction product was visualized by the addition of diaminobenzidine substrate (Vector Lab.). Sections were counterstained with hematoxylin. Negative controls were obtained by incubating sections with phosphate-buffered saline (PBS) instead of primary antibodies.
qRT-PCR. Total RNA was isolated from testes of adult rats using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to manufacturer's instructions. The qRT-PCR assays were performed using oligonucleotide primers for 18S rRNA (5′-ACACGGACAGGATTGACAGATT; 5′-CGTTCGTTATCGGAATTAACCA) and rat IDO (5′-AGCACTGGAGAAGGCACTG and 5′-CGTGGAAAAAGGTGTCTGG). 18S rRNA was chosen as housekeeping gene. Reactions were conducted using FastStart Universal SYBR Green Master Mix (Roche Diagnostic, Mannheim, Germany) and the CFX-96 touch sequence detector System (Bio-Rad, Hercules, CA, USA). Reaction conditions were as follows: 10 min at 95 °C (one cycle), followed by 40 cycles of 15 s at 95 °C, 30 s at 55 °C and 1 min at 60 °C for 18S, or by 40 cycles of 15 s at 95 °C and 1 min at 60 °C for IDO. Following the mathematical model of Pfaffl 30 , the fold change of mRNA IDO expression was determined for each sample as described previously 31 .

Immunohistochemical analysis followed by laser capture microdissection and RT-PCR. A goat
polyclonal anti-vimentin antibody (1/10, Santa Cruz Biotechnology, Dallas, Texas, USA) was used to identify Sertoli cells in testes paraffin embedded sections. An immunoperoxidase technique using the avidin-biotin system was applied as described above. Subsequently, laser capture microdissection (LCM) was performed. Laser energy of the Applied Biosystems (Bedford, MA, USA) Arcturus LCM equipment was used to circumscribe vimentin immunopositive Sertoli cells as previously described by Rossi 31 . Microdissected samples containing immunopositive Sertoli cells were used for RT-PCR. Total RNA was isolated using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. Purity of total RNA isolated was assessed using specific markers of germ cells (VASA) and Sertoli cells (FSH-receptor). The following oligonucleotide primers were used for amplification of IDO (first set 5′-GCAGTAGAGCATCAAGACC and 5′-CGTGGAAAAAGGTGTCTGG, hemi-nested set 5′-AGCACTGGAGAAGGCACTG and 5′-CGTGGAAAAAGGTGTCTGG), VASA (5′-CCAAGAGAGGCGGCTATCGAGATG and 5′-AGAACCAAAAAGGCCAACCAGTGCG), and FSH-receptor (5′-CCTTGCTCCTGGTCTCCTTG and 5-GGAAGACCCTGTCAGAGC). 18S amplification was performed using the oligonucleotide primers previously described in qRT-PCR section. PCR conditions were 95 °C for 5 min, followed by 30-45 cycles of 94 °C for 1 min, 60 °C (annealing temperature) for 1 min, 72 °C for 1 min, and a final incubation at 72 °C for 5 min. PCR products were separated on 2% agarose gels and visualized with ethidium bromide.
Western blots. Expression of IDO and TDO proteins were evaluated in testis, in a pool of renal and iliac LN (testicular draining LN; TLN) and a pool of inguinal and popliteal LN (LN draining the site of immunization, ILN). ST were mechanically separated from testicular interstitial cell (IC) and homogenized in ice-cold lysis buffer as described previously 32 . LN were cut with scissors and incubated with the lysis buffer. 200 μg of proteins were resolved in a 10% SDS-polyacrylamide gel electrophoresis, electroblotted at 66 mA for 18 h onto PVDF membranes (Bio-Rad Laboratories) and transfer was monitored by Ponceau S staining. Membranes were blocked with 5% non-fat dry milk in TBS containing 0.1% Tween 20 for 1 h. Blots were probed overnight with a rabbit anti-IDO 29 (1/100), rabbit anti-IDO2 (1/100) or anti-TDO (1/500, GeneTex) polyclonal antibody followed by anti-actin (1/6000; Sigma-Aldrich) polyclonal antibody. Blots were washed and incubated with a biotinylated goat anti-rabbit IgG (1/3000; Vector Lab.) followed by streptavidin-horseradish peroxidase conjugates (Chemicon International Inc, Millipore Co., Billerica, MA, USA). Proteins were visualized by enhanced chemiluminescence and images were captured using GeneSnap software (7.08.01 version) and analyzed with Gene Tools software (4.01.02 version) from SynGene (Synoptics Ltd, Frederick, MD, USA).

IDO/TDO activity assay.
To detect IDO/TDO activity decapsulated testes, TLN and ILN were cut with scissors. Samples were incubated with lysis buffer as described above and no protease inhibitors were added. Protein precipitation was performed by adding 70% perchloric acid to the supernatant. The suspension was left on ice for 15 min, and then centrifuged at 22,000 g for 10 min. The pellet was discarded and 100 μl of the supernatant was analyzed by HPLC. Kynurenine (Kyn) release and tryptophan (Trp) degradation were measured. Standard curves were generated with L-Kyn and L-Trp (Sigma-Aldrich) in the same solvent.

Administration of 1-Methyl-Tryptophan (1-MT).
Based on Hou and Muller 33 , we used IDO specific inhibitor 1-D-MT (Sigma-Aldrich). It was administered 5 days per week by oral gavage at 10 mg/dose (0.1 ml/ rat) during the immunization period (from day 0 to day 28) to rats immunized with TH and adjuvants. Another group of immunized rats received 0.1 ml of diluent solution (0.5% Methylcellulose, 0.5% Tween 80) (vehicle). Animals were killed 50 days after first immunization. Body weight was determined and testes and epididymis were removed and processed for histopathology.
Delayed type hypersensitivity (DTH). DTH was measured by a footpad swelling test performed at the end of the in vivo assay as previously described 34 . Statistical analysis. Results are expressed as mean ± SEM. Comparisons between groups were assessed by the non-parametric Mann-Whitney U test or Kruskal-Wallis One-Way ANOVA. P values less than 0.05 were considered significant.

Results
Histopathology. As described previously 26 rats immunized with TH and adjuvants developed autoimmune orchitis characterized by mild interstitial infiltration of lymphomononuclear cells intermingled with Leydig cells and multiple foci of damaged ST with different degrees of germ cell sloughing. Some rats with severe orchitis presented severe germ cell sloughing in most ST in which only SC and basal germ cells (mainly spermatogonia and few pre leptotene spermatocytes) remained attached to the tubular wall and granulomae. mRNA expression of IDO decreased in whole testes and isolated SC during EAO. To understand the relevance of IDO in testicular immune-privilege, we first compared IDO mRNA expression of N testis with IDO mRNA levels in testis from C and EAO groups. By qRT-PCR, we observed that testicular IDO expression was down regulated in rats with orchitis compared to C and N groups. Testis from C and N groups presented similar IDO mRNA expression levels (Fig. 1).
Based on the fact that SC are considered the paradigmatic testicular somatic cells with an immune regulatory role, we evaluated IDO mRNA expression in SC isolated from N and EAO groups. LCM followed by RT-PCR analyses revealed visible PCR products only in SC from N testis (Fig. 2). In addition, expression of germ cell marker (VASA) was not seen in isolated SC (Fig. S1).
Expression and localization of IDO and TDO. Protein IDO1 expression was evaluated by Western blot analysis in testicular ST and IC fractions from N, C, and EAO groups. IDO content in the ST fraction isolated from EAO rats was significantly lower compared to N and C rats (Figs 3A and S2A). No significant differences in IDO1 content were observed in the IC fraction in the different groups studied (Figs 3B and S2B).
In order to examine which cell populations of the testis might be contributing to IDO1 expression, immunohistochemistry was performed on testicular sections of N, C, and EAO groups. Intensity of IDO staining was similar in N ( Fig. 4A-C (data not shown) rats. Positive staining was observed in the testicular interstitium of the three groups studied, localized mainly in the endothelium of blood vessels (Figs 4 and S3). In EAO rats mild IDO reactivity was also detected in mononuclear cells present in the testicular interstitium, identified as resident macrophages (ED2+ cells) and DC and inflammatory macrophages (ED1+ cells) (Fig. S3). Exceptionally, specific staining was detected in spermatid heads of ST (stages XI and XIV) from testes of N and C rats (data not shown).
Considering that TDO and IDO2 also catalyze the initial step of tryptophan catabolism and TDO and IDO2 mRNA are expressed in testis 35,38 , we evaluated protein TDO and IDO2 expression. No significant differences in TDO and IDO2 content were detected by Western blot analysis in testis from N, C, and EAO groups (Figs 5 and  S4). www.nature.com/scientificreports www.nature.com/scientificreports/ TDO localization was evaluated by indirect immunoperoxidase in testicular sections of N, C, and EAO groups. TDO reactivity was detected in cells with macrophage/dendritic morphology present in granulomas and in testicular interstitium from EAO rats (Fig. 6A,B). No staining was observed in sections of N (Fig. 6C) and C rats (data not shown).

IDO/TDO activity in testis. IDO/TDO activity was assessed by HPLC to measure concentrations of Trp
and Kyn in supernatants collected from testicular and LN homogenates. IDO activity (Kyn/Trp ratio) was reduced in testis of the EAO group compared to N and C groups (Fig. 7).

In vivo inhibition of IDO increased severity of EAO.
To elucidate the role of IDO in testicular immune regulation, we evaluated the impact of inhibiting IDO during EAO induction. As described in Materials and Methods, immunized rats were treated or not with a specific enzyme inhibitor 1-MT until euthanasia. Notably, rats treated with 1-MT during the immunization period developed orchitis with significantly increased severity (Fig. 8B). Testicular histopathology showed that vehicle group presented multifocal testicular damage characterized by mild infiltration of mononuclear cells and foci of ST with different degrees of germ cell sloughing. In contrast, most rats treated with 1-MT presented extended areas of severely damaged ST presenting aspermatogenesis (Fig. 8A).
Epididymis of EAO rats treated with 1-MT compared to EAO vehicle group showed a higher degree of inflammation represented by interstitial immune cell infiltrates and similar sperm depletion in the tubular lumen (Fig. 8A). However quantitative data of epididymal pathology were not statistically different (Fig. 8B).
Body weight of rats treated with 1-MT did not differ from that of untreated rats suggesting that no apparent detrimental effects were induced by IDO inhibition treatments (Body weight (g) mean ± SEM: 1-MT, 464.00 ± 9.73; vehicle group, 488.90 ± 9.40).
We subsequently investigated effects of IDO inhibition on T cell mediated immunity by analyzing DTH response to testicular antigens. Footpad challenge for DTH was tested in rats treated with 1-MT or vehicle 50 days after immunization. No significant differences in footpad swelling were observed between 1-MT and vehicle (Fig. S5). These results showed that both experimental groups reacted similarly to the antigen challenge independently of 1-MT treatment.

Evaluation of IDO and TDO in lymph nodes.
Since draining LN reflect immune cell microenvironment of target organ, IDO1 and TDO expression was also evaluated in testicular draining LN and non-draining LN. Intense IDO staining was observed mainly in cells with macrophage/dendritic morphology present in the deep cortex and in the medullary cords of draining and non-draining LN. Endothelial cells from blood vessels also exhibited a slightly positive staining for IDO. The highest number of positive IDO cells was observed in www.nature.com/scientificreports www.nature.com/scientificreports/ draining LN of EAO rats. Also, a higher number of IDO positive cells were observed in draining LN compared to non-draining LN (Fig. 9A). LN of N rats presented similar IDO expression compared to rats in C group (data not shown).
No significant differences in IDO and TDO content were detected comparing N, C, and EAO rats in lysates of draining and non-draining LN (Figs 9B and S6).
Regarding IDO/TDO activity in LN, no significant changes were detected in draining and non-draining LN of EAO rats compared to N and C groups (Fig. 10).

Discussion
Although tryptophan catabolism into kynurenine by the IDO enzyme has been proposed as a putative mechanism in testicular immune tolerance, this possibility has never been evaluated in normal and pathological conditions in rats. This hypothesis stimulated us to evaluate the role of IDO in testis homeostasis and inflammation (autoimmune orchitis) in which immune privilege is disrupted. We evaluated the expression and activity of IDO/ TDO in normal versus inflamed testis and assessed orchitis progression after treating rats immunized with sperm antigens and adjuvants with the IDO-specific inhibitor 1MT.
In the male reproductive tract, it has been described a null 35,36 or negligible IDO mRNA and protein expression in the testis of mice 37 , contrasting with its high content in epididymis. More recently Jenabian et al. 38 determined IDO1 and IDO2 mRNA in human testis of antiretroviral therapy-treated HIV-infected and non infected individuals. Our data show that rat testes with normal histopathology in the normal and control groups www.nature.com/scientificreports www.nature.com/scientificreports/ expressed IDO mRNA and IDO1 protein as analyzed by Western blot. By immunohistochemistry we observed that IDO1 + cells were found in the testicular interstitium mainly in endothelial cells, and also in mononuclear cells, as described for other organs 39,40 . The fact that we did not detect IDO1 in SC by immunohistochemistry but did identify IDO mRNA in isolated SC and IDO protein in seminiferous tubules by Western blot might be due to the different sensitivity of techniques. Prepuberal SC isolated from normal pig have been demonstrated to progressively produce IDO and prolong survival of co-grafts in different immunological settings 9,41 . As a relevant result, we observed IDO mRNA in SC from testis of normal untreated rats only but not in SC from rats with autoimmune orchitis. Notably, IDO mRNA and protein expression as well as IDO activity were reduced in testis from EAO rats, suggesting that testis inflammation is associated with downregulation of IDO expression and enzyme activity.
IFNγ produced in inflamed tissues is known to be potent IDO inducer because mammalian ido1 gene promoters contain IFN-stimulated response elements 42 . We previously reported the increase of IFNγ in conditioned medium of testicular macrophages 27 and in the number of IFNγ-producing CD4+ T cells in rats with orchitis 43 . However, present results do not reflect positive regulation of IDO by IFN. Also, ido1 gene transcription may not result in enhanced IDO enzyme activity due to posttranslational controls, such as nitric oxide (NO) 44 . We may speculate that in EAO, www.nature.com/scientificreports www.nature.com/scientificreports/ NO-IDO interaction limits IDO function considering that testicular NO increases in rats with autoimmune orchitis 45 and prevention of EAO development occurs by inhibition of the nitric oxide synthase-NO system 46 . Similarly, this interaction was also demonstrated in heart allografts in rats 47 and in a model of collagen-induced arthritis 48 .   www.nature.com/scientificreports www.nature.com/scientificreports/ Testicular TDO content was also similar among different groups studied. Notably, in few rats with severe orchitis; we observed TDO expression in macrophages and dendritic-like cells in the periphery of granulomae, which wall off immunogenic apoptotic germ cells from other structures of the tissue. By immunohistochemistry, Britan et al. 35 detected that TDO protein was present both in the intertubular tissue and in late differentiating germ cells of adult mouse testis Finally, to elucidate the role of IDO in testicular immune tolerance in vivo, we evaluated the impact of the specific IDO inhibitor 1-MT administered during the immunization period, demonstrating that IDO inhibition increased the severity of EAO. Therefore tryptophan metabolism could play a critical role in homeostatic control of testicular immune privilege. Since EAO is associated with epididymitis 26,32 , and the absence of IDO generates inflammation in the caput epididymis 53 , we also evaluated epididymis. IDO inhibition had no impact on epididymis histopathology. By analyzing in vivo DTH response to spermatic antigens, we showed that 1-MT treatment did not modify cell mediated immunity, thereby strengthening the hypothesis of a local mechanism of testicular IDO function as mentioned above in reference to LN results. Although they used a different experimental design of 1-MT administration, early results of Sakurai et al. 54 and Kwidzinski et al. 55 also showed that IDO inhibition exacerbates the clinical course of EAE, which supports a role for IDO in controlling T cell-mediated inflammation. Rats from vehicle group presented focal testicular damage characterized by a moderate interstitial mononuclear cell infiltrate and several seminiferous tubules with different degrees of germ cell sloughing. In contrast, rats from 1-MT group presented severe orchitis with large areas of aspermatogenic seminiferous tubules showing only spermatogonia and Sertoli cells attached to the tubular wall. Epididymal histopathology of rats from 1-MT group compared to vehicle group shows similar depletion of spermatozoa and increase of inflammatory cell infiltrate (*). H&E. (B) Severity of orchitis and epididymitis is expressed as score mean ± SEM of vehicle and 1-MT group rats. Figure 9. Evaluation of IDO1 and TDO in lymph nodes (LN). (A) Photomicrographs of testicular draining and non draining LN cryostat sections immunostained with specific IDO antibody. IDO immunoreactivity can be observed in endothelial cells of blood vessels (asterisk) and cells with macrophage/dendritic morphology (arrowhead). Note a higher number of positive IDO cells in draining LN from EAO rats. Omission of primary anti-IDO1 antibody was used as a negative control. (B) Expression of IDO and TDO in LN of normal, control, and EAO rats was analyzed by Western blot. Each bar represents the mean ± SEM of 6 animals. The blots were cropped and the full-length blots are presented in the supplementary information (Fig. S6).